The first goal of this project is to define the roles of fibroblast growth signalling in pregastrulation mouse development. The second goal is to define the role of ICM-derived FGF-4 in this developmental period. FGFs are crucial for gastrulation in frog; overexpression leads to increased posterior mesoderm and ablation of FGF signalling leads to loss of posterior mesoderm. Similarly in mouse a null mutant for a single FGF ligand (FGF-3), has a deficit of posterior mesoderm. FGF-4 is unique among the growth factors studied in pregastrulation mouse embryos in its restriction th the inner cell mass (ICM). Since the ICM is the precursor to all embryonic cell layers as well as extraembryonic endoderm and mesoderm, ICM-derived FGF-4 may be important in mediating early events in mouse growth and differentiation between blastocyst-stage and gastrulation. Overexpression of FGF-4 increases the number of endoderm growing out from the ICM. Transcripts for all 4 high affinity receptors for FGF and a low affinity receptor (syndecan-1) are expressed in blastocyst and in blastocyst outgrowths. FGFR-3 and FGFR-4 are expressed at the highest levels. The first goal is to ablate all FGF signalling in the mouse embryo through the blastocyst stage and test for effects. The second goal is to overexpress and underexpress FGF-4 ligand in mouse embryos through the blastocyst stage. This will give information on the importance of FGF-4 in early development and may indicate the affected cells and stages at which the effects occur. Expression vectors for a dominant negative FGF receptor (dnFGFR), for coding sequence FGF-4 and antisense FGF-4 have been constructed. The promoter used will insure expression in every cell of the blastocyst after microinjection into the male pronucleus (integration is not required). The modality of delivery of dnFGFR and assay of effects of dnFGFR (or of FGF-4 sense or antisense) will be determined after initial experiments. Modes of delivery include microinjection into the oocyte, or one blastomere in the 4 cell embryo (to access lineage of descendant cells) or blastocyst injection of stably transfected embryonic stem cells (if these remain totipotent). The later two modes allow lineage tracing in the case of early lethality. Modes of assay include assay for viability (of blastocyst, gastrulation-stage and day 11.5 p.c. embryo), assay of gross morphology of day 11.5 p.c. embryo, molecular phenotype (expressions of brachyury at gastrulation), and assay of phenotypes (of cell/morphology) in ICM and blastocyst outgrowths and in gastrulating and 11.5 day p.c. embryos. Experimentally, the goal will be to first find the largest (morphological) phenotypes and then refine these to yield cellular and molecular information. If lethality occurs early, then lineage tracing experiments will be assayed. Later studies may include the use of alternate or inducible promoters to target the effects of dnFGFR or of the coding FGF-4 to specific embryonic tissues or times. Since the events studied occur during an important teratogenic period these studies may lead to an improved understanding of the etiology of teratogenesis.
| Patel, Y; Kim, H; Rappolee, D A (2000) A role for hepatocyte growth factor during early postimplantation growth of the placental lineage in mice. Biol Reprod 62:904-12 |
| Chai, N; Patel, Y; Jacobson, K et al. (1998) FGF is an essential regulator of the fifth cell division in preimplantation mouse embryos. Dev Biol 198:105-15 |
| Rappolee, D A; Iyer, A; Patel, Y (1996) Hepatocyte growth factor and its receptor are expressed in cardiac myocytes during early cardiogenesis. Circ Res 78:1028-36 |
